: Leigh?s Syndrome (LS) is a disease that arises due to mutations in the mitochondrial genome, leading to neuronal and neuromuscular dysfunction. The disease severity is influenced by the percentage of mutant versus healthy mtDNA, but cellular effects and clinical symptoms can be highly variable. There is currently no cure for LS and the lack of quantitative functional biomarkers for LS impedes the discovery and testing of novel therapies. To this end, we aim to utilize label-free multiphoton microscopy to establish endogenous optical biomarkers of metabolic dysfunction associated with LS. We will exploit the natural fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), which are metabolic cofactors that play a key role in linking oxidative phosphorylation and glucose catabolism. Our central hypothesis is that this natural fluorescence coming from mitochondria can provide a sensitive measure of metabolic dysfunction following point mutations in mitochondrial DNA. Our multidisciplinary team will build upon established expertise in label-free multiphoton microscopy, mitochondrial genetics and physiology, and stem cell engineering. We have obtained LS patient-derived fibroblasts from previous work, and in Aim 1, we will reprogram and differentiate these cells into neurons and myocytes given that LS primarily affects neurological and neuromuscular function. Cell lines from normal and LS patients will be imaged using a label-free multiphoton microscopy, and an optical redox ratio of FAD/(NADH+FAD) fluorescence intensity, NADH fluorescence lifetime, and mitochondrial organization will be compared among groups. In Aim 2, we will evaluate whether we can identify distinct point mutations that affect Complex I or Complex V of the electron transport chain through dynamic changes in our biomarkers in response to rotenone and oligomycin. It is anticipated that the development of endogenous optical biomarkers of LS through non-invasive label-free multiphoton imaging techniques would provide immediate directions for improving the diagnosis of mitochondrial disorders in vivo and establishing quantitative parameters for an in vitro drug testing platform.